Water soluble fluorescent or colored dyes and methods for their use

ABSTRACT

Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I): 
                         
including stereoisomers, salts and tautomers thereof, wherein R 1a , R 1b , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i , R 2j , R 2k , R 2l , R 2m , R 2n , R 2o , R 2p , R 2q , R 2r  and R 2s  are as defined herein. Methods associated with preparation and use of such compounds are also provided.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention is directed to novel fluorescent or colored dyesand methods for their preparation and use in various analytical methods.

Description of the Related Art

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, saccharides, pharmaceuticals, metabolites,microorganisms, ions, and other materials of diagnostic value. Examplesof such materials include narcotics and poisons, drugs administered fortherapeutic purposes, hormones, pathogenic microorganisms and viruses,peptides, e.g., antibodies and enzymes, and nucleic acids, particularlythose implicated in disease states.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity that characterizesmany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques, and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which is attached toone or more of the interacting materials. The specific labeling methodchosen often dictates the usefulness and versatility of a particularsystem for detecting an analyte of interest. Preferred labels areinexpensive, safe, and capable of being attached efficiently to a widevariety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding affinities of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations. However, suchlabels are expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance, and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest because of the large number of such labels that areknown in the art. Moreover, the literature is replete with syntheses offluorescent labels that are derivatized to allow their attachment toother molecules, and many such fluorescent labels are commerciallyavailable.

Cyanine dyes have been widely used for labeling biomolecules includingantibodies, DNA probes, avidin, streptavidin, lipids, biochemicalanalogs, peptides, and drugs, as well as for a variety of applicationsincluding DNA sequencing, DNA microarray, western blotting, flowcytometry analysis, and protein microarrays, to name a few. Scientistsfavor using cyanine dyes in biological applications because, among otherreasons, cyanine dyes 1) are biocompatible; 2) have high molarabsorptivity (c.a. 10⁵ M⁻¹ cm⁻¹); 3) are readily modified to match awide range of desired excitation and detection wavelengths (e.g., about500 to about 900 nm); 4) are capable of incorporating water-solublegroups and linking groups; 5) and possess favorable fluorescenceproperties. In particular, Cy2 conjugates, with a maximumadsorption/excitation around 492 nm and emission around 510 nm, in thegreen region of the visible spectrum, are commonly used as analternative to FITC due to reduced sensitivity to pH changes. However,the low fluorescence quantum yield, short fluorescence lifetime,propensity to photobleach, and poor chemical stability of Cy2 haslimited its use in chemical and life sciences.

There is thus a need in the art for water soluble dyes and biomarkersthat permit visual or fluorescent detection of biomolecules withoutprior illumination or chemical or enzymatic activation. Ideally, suchdyes and biomarkers should be intensely colored or fluorescent andshould be available in a variety of colors and fluorescent wavelengths.The present invention fulfills this need and provides further relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

In brief, the present invention is generally directed to compoundsuseful as water soluble, fluorescent or colored dyes and probes thatenable visual detection of biomolecules and other analytes, as well asreagents for their preparation. Methods for visually detecting abiomolecule and for determining the size of a biomolecule are alsodescribed. The water soluble, fluorescent or colored dyes of theinvention are intensely colored and/or fluorescent and can be readilyobserved by visual inspection or other means. In some embodiments thecompounds may be observed without prior illumination or chemical orenzymatic activation. Advantageously, embodiments of the dyes have amaximum absorbance ranging from about 468 nm to about 508 nm and amaximum emission ranging from about 495 nm to about 525 nm. For example,in certain embodiment the dyes have a maximum absorbance at about 490 nmand a maximum emission at about 505 nm. The dyes are thus ideal for usein various analytical methods. By appropriate selection of the dye, asdescribed herein, visually detectable biomolecules of a variety ofcolors may be obtained.

Accordingly, in one embodiment a compound having the following structure(I) is provided:

or a salt, stereoisomer or tautomer thereof, wherein R^(1a), R^(1b),R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r)and R^(2s) are as defined herein.

In other embodiments, an analyte molecule comprising a covalent bond toa compound having the following structure (I′):

or a salt, stereoisomer or tautomer thereof is provided, wherein R^(1a),R^(1b), R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h),R^(2i), R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q),R^(2r) and R^(2s) are as defined herein and wherein at least one ofR^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r)and R^(2s) is the analyte molecule or a linkage thereto.

In another embodiment, a method for staining a sample is provided; themethod comprises adding to said sample a representative compound asdescribed herein in an amount sufficient to produce an optical responsewhen said sample is illuminated at an appropriate wavelength.

In still other embodiments, the present disclosure provides a method forvisually detecting a biomolecule, comprising:

(a) providing a representative compound described herein; and

(b) detecting the compound by its visible properties.

Other disclosed methods include a method for visually detecting abiomolecule, the method comprising:

(a) admixing any of the disclosed compounds with one or morebiomolecules; and

(b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising any one ofthe disclosed compounds and one or more biomolecules. Use of suchcomposition in analytical methods for detection of the one or morebiomolecules is also provided.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the ¹H NMR spectrum of compound 2 in CDCl₃.

FIG. 2 depicts a representative total diode array chromatogram (215-500nm) of a 2 μL injection volume of compound 2. System used was a WatersAcquity UHPLC system with a 2.1 mm×50 mm Acquity BEH-C18 column held at45° C.

FIG. 3 depicts the background-subtracted mass spectrum of brominatedanthracene derivative 2 product. Expected mass: 617.8.

FIG. 4 depicts the ¹H NMR spectrum of compound 3 in CDCl₃.

FIG. 5 depicts a representative total diode array chromatogram (215-500nm) of a 2 μL injection volume of compound 3. System used was a WatersAcquity UHPLC system with a 2.1 mm×50 mm Acquity BEH-C18 column held at45° C.

FIG. 6 depicts the background-subtracted mass spectrum of compound 3.Expected mass 929.1.

FIG. 7 depicts a representative chromatogram at 488 nm of a 10 μLinjection volume of compound 5 (crude 5′-NH3-(HEG)-AQ6-(HEG)-3′sequence). System used was a Waters Acquity UHPLC system with a 2.1mm×50 mm Acquity BEH-C18 column held at 45° C.

FIG. 8 depicts the background-subtracted mass spectrum of compound 5(crude 5′-NH3-(HEG)-AQ6-(HEG)-3′ sequence). The method employselectrospray ionization in negative mode and shows ⁻1, ⁻2, and ⁻3 chargestates.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ group.

“Carboxy” refers to the —CO₂H group.

“Cyano” refers to the —CN group.

“Formyl” refers to the —C(═O)H group.

“Hydroxy” or “hydroxyl” refers to the —OH group.

“Imino” refers to the ═NH group.

“Nitro” refers to the —NO₂ group.

“Oxo” refers to the ═O substituent group.

“Sulfhydryl” refers to the —SH group.

“Thioxo” refers to the ═S group.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Unless stated otherwise specifically in the specification, analkyl group is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a substituentgroup, consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to thesubstituent group through a single or double bond. The points ofattachment of the alkylene chain to the rest of the molecule and to thesubstituent group can be through one carbon or any two carbons withinthe chain. Unless stated otherwise specifically in the specification, analkylene chain is optionally substituted.

“Alkoxy” refers to a group of the formula —OR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms. A“hydroxylalkoxy” is an alkoxy moiety comprising at least one hydroxylsubstituent. An “aminoalkoxy” is an alkoxy moiety comprising at leastone amino substituent. Unless stated otherwise specifically in thespecification, alkoxy, hydroxylalkoxy and/or aminoalkoxy groups areoptionally substituted.

“Alkylamino” refers to a group of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl group as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group is optionallysubstituted.

“Alkylether” refers to any alkyl group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylethersinclude at least one carbon oxygen bond, but may include more than one(i.e., a “polyalkylether”). For example, polyethylene glycol (PEG),which is a polyalkylether, is included within the meaning of alkylether.“Hydroxylpolyalkylether” refers to a polyalkylether comprising one ormore hydroxyl substituents. “Aminopolyalkylether” refers to apolyalkylether comprising one or more amino substituents. Unless statedotherwise specifically in the specification, an alkylether,polyalkylether, hydroxylpolyalkylether and/or aminopolyalkylether groupis optionally substituted.

“Alkylenether” refers to any alkylene group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylenethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylenether. Unless stated otherwise specifically in the specification,an alkylenether group is optionally substituted.

“Alkylphospho” refers to the —RP(═O)(R_(a))R_(b) group, wherein R is analkylene group, R_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkyl or—Oalkylether, wherein R_(c) is a counter ion (e.g., Na+ and the like).Unless stated otherwise specifically in the specification, analkylphospho group may be optionally substituted. For example, incertain embodiments, the alkyl or alkylether moiety (R_(b)) in analkylphospho group is optionally substituted with one or more ofhydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whichsubstituents are al so optionally substituted.

“Oalkylphospho” is an alkylphospho group connected to the remainder ofthe molecule via an oxygen atom. Unless stated otherwise specifically inthe specification, an Oalkylphospho group is optionally substituted. Forexample, in certain embodiments, the alkyl, alkylether or polyalkylethermoiety (R_(b)) in an Oalkylphospho group is optionally substituted withone or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether, which substituents are also optionallysubstituted.

“Alkyetherphospho” refers to the —RP(═O)(R_(a))R_(b) group, wherein R isan alkylenether group, R_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylor —Oalkylether, wherein R_(c) is a counter ion (e.g., Na+ and thelike). Unless stated otherwise specifically in the specification, analkyletherphopsho group is optionally substituted. For example, incertain embodiments, the alkyl or alkylether moiety (R_(b)) in analkyletherphospho group is optionally substituted with one or more ofhydroxyl, amino sulfhydryl or a phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whichsubstituents are al so optionally substituted.

“Oalkyletherphospho” is an alkyletherphospho group connected to theremainder of the molecule via an oxygen atom. Unless stated otherwisespecifically in the specification, an Oalkyletherphospho group isoptionally substituted. For example, in certain embodiments, the alkylor alkylether moiety (R_(b)) in an Oalkyletherphospho group isoptionally substituted with one or more of hydroxyl, amino sulfhydryl ora phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents are al sooptionally substituted.

“Alkylthiophospho” refers to the —RP(═R_(a))(R_(b))R_(c) group, whereinR is an alkylene group, R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) orSR_(d); and R_(c) is —Oalkyl or —Oalkylether, wherein R_(d) is a counterion (e.g., Na+ and the like) and provided that: R_(a) is S or R_(b) isS⁻ or SR_(d); or provided that R_(a) is S and R_(b) is S⁻ or SR_(d).Unless stated otherwise specifically in the specification, aalkylthiophospho group is optionally substituted. For example, incertain embodiments, the alkyl or alkylether moiety in a alkythiophosphogroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents are alsooptionally substituted.

“Oalkylthiophospho” is an alkylthiophospho group connected to theremainder of the molecule via an oxygen atom. Unless stated otherwisespecifically in the specification, an Oalkylthiophospho group isoptionally substituted. For example, in certain embodiments, the alkylor alkylether moiety in an Oalkythiophospho group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether, which substituents are also optionallysubstituted.

“Alkyletherthiophospho” refers to the —RP(═R_(a))(R_(b))R_(c) group,wherein R is an alkylenether group, R_(a) is O or S, R_(b) is OH, O⁻,S⁻, OR_(d) or SR_(d); and R_(c) is —Oalkyl or —Oalkylether, whereinR_(d) is a counter ion (e.g., Na+ and the like) and provided that: R_(a)is S or R_(b) is S⁻ or SR_(d); or provided that R_(a) is S and R_(b) isS⁻ or SR_(d). Unless stated otherwise specifically in the specification,an alkyletherthiophospho group is optionally substituted. For example,in certain embodiments, the alkyl or alkylether moiety in aalkyletherthiophospho group is optionally substituted with one or moreof hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whichsubstituents are also optionally substituted.

“Oalkyletherthiophospho” is an alkyletherthiophospho group connected tothe remainder of the molecule via an oxygen atom. Unless statedotherwise specifically in the specification, an Oalkyletherthiophosphogroup may be optionally substituted. For example, in certainembodiments, the alkyl or alkylether moiety in an Oalkyletherthiophosphogroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents are alsooptionally substituted.

“Amidyl” refers to the —NR_(a)R_(b) radical, wherein R_(a) and R_(b) areindependently H, alkyl or aryl. Unless stated otherwise specifically inthe specification, an amide group is optionally substituted.

“Aryl” refers to a hydrocarbon ring system group comprising hydrogen, 6to 18 carbon atoms and at least one aromatic ring. For purposes of thisinvention, the aryl group may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl groups include, but are not limited to, aryl groupsderived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl groups thatare optionally substituted.

“Aryloxy” refers to a group of the formula —OR_(a), where R_(a) is anaryl moiety as defined above, for example phenoxy and the like. Unlessstated otherwise specifically in the specification, an aryloxy group isoptionally substituted.

“Aralkyl” refers to a group of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more aryl groupsas defined above, for example, benzyl, diphenylmethyl and the like.Unless stated otherwise specifically in the specification, an aralkylgroup is optionally substituted.

“Oaralkyl” is an aralkyl group which is connected to the remainder ofthe molecule via an oxygen linkage. “ODMT” refers to dimethoxytrityllinked to the rest of the molecule via an O atom. Unless statedotherwise specifically in the specification, an Oaralkyl group isoptionally substituted.

“Cyanoalkyl” refers to an alkyl group comprising at least one cyanosubstituent. The one or more —CN substituents may be on a primary,secondary or tertiary carbon atom. Unless stated otherwise specificallyin the specification, a cyanoalkyl group is optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon group consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic cycloalkylgroups include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groupsinclude, for example, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwisestated specifically in the specification, a cycloalkyl group isoptionally substituted.

“Cycloalkylalkyl” refers to a group of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylgroup as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group is optionally substituted.

“Multicyclic” refers to any molecule having more than one ring. Therings may be either, fused, spirocyclic or separated by one or moreatoms (e.g., linked via an acyclic linker).

“Spirocyclic” refers to a multicyclic molecule wherein two rings share asingle carbon atom.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl group, as defined above, that issubstituted by one or more halo groups, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group is optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring group which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl group may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl group may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclyl groupmay be partially or fully saturated. Examples of such heterocyclylgroups include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, a heterocyclyl group is optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl group as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl group to the rest of the molecule is through a nitrogenatom in the heterocyclyl group. Unless stated otherwise specifically inthe specification, a N-heterocyclyl group is optionally substituted.

“Heterocyclylalkyl” refers to a group of the formula —R_(b)R_(e) whereR_(b) is an alkylene chain as defined above and R_(e) is a heterocyclylgroup as defined above, and if the heterocyclyl is a nitrogen-containingheterocyclyl, the heterocyclyl may be attached to the alkyl group at thenitrogen atom. Unless stated otherwise specifically in thespecification, a heterocyclylalkyl group is optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system group comprisinghydrogen atoms, one to thirteen carbon atoms, one to six heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur, andat least one aromatic ring. For purposes of this invention, theheteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclicring system, which may include fused or bridged ring systems; and thenitrogen, carbon or sulfur atoms in the heteroaryl group may beoptionally oxidized; the nitrogen atom may be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl,benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxi dopyrazinyl, 1-oxidopyri dazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group is optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl group as defined above containingat least one nitrogen and where the point of attachment of theheteroaryl group to the rest of the molecule is through a nitrogen atomin the heteroaryl group. Unless stated otherwise specifically in thespecification, an N-heteroaryl group is optionally substituted.

“Heteroarylalkyl” refers to a group of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and R_(f) is a heteroarylgroup as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group is optionally substituted.

“Hydroxylalkyl” refers to an alkyl group comprising at least onehydroxyl substituent. The one or more —OH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, hydroxyalkyl group is optionallysubstituted.

“Hydroxylalkylether” refers to an alkylether group comprising at leastone hydroxyl substituent. The one or more OH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, hydroxyalkylether group is optionallysubstituted.

“Phosphate” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a further phosphate group(as in diphosphate and triphosphate) thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(c) is a counter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphate group is optionallysubstituted.

“Phospho” refers to the —P(═O)(R_(a))R_(b) group, wherein R_(a) is OH,O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a phosphate group (as indiphosphate and triphosphate) thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(c) is a counter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phospho group is optionallysubstituted.

“Phosphoalkyl” refers to the —P(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is —Oalkyl, wherein R_(c) is a counter ion(e.g., Na+ and the like). Unless stated otherwise specifically in thespecification, a phosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the alkyl moiety in a phosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl or a phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whichsubstituent is optionally substituted.

“Ophosphoalkyl” is a phosphoalkyl group connected to the remainder ofthe molecule via an oxygen atom. Unless stated otherwise specifically inthe specification, an Ophosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the alkyl moiety in an Ophosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl or a phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whichsubstituent is optionally substituted.

“Phosphoalkylether” refers to the —P(═O)(R_(a))R_(b) group, whereinR_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylether (including polyethers such as polyethyleneoxide ethers and the like), wherein R_(c) isa counter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphoalkylether group isoptionally substituted. For example, in certain embodiments, thealkylether moiety in a phosphoalkylether group is optionally substitutedwith one or more of hydroxyl, amino, sulfhydryl, phosphate, phospho,thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents areoptionally substituted.

“Ophosphoalkylether” is a phosphoalkylether group connected to theremainder of the molecule via an oxygen atom. Unless stated otherwisespecifically in the specification, an ophosphoalkylether group isoptionally substituted. For example, in certain embodiments, thealkylether moiety in an Ophosphoalkylether group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,phospho, thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents areoptionally substituted.

“Phosphoramidite” refers to the —OP(OR^(a))(NR^(b) ₂) group, whereinR^(a) is alkyl and each R^(b) is independently H or alkyl. Unless statedotherwise specifically in the specification, a phosphoramidite group isoptionally substituted.

“Activated phosphorous” refers to any moiety comprising phosphorouswhich is cable of reaction with a nucleophile, for example reacting witha nucleophile at the phosphorous atom. For example, phosphoramidites andmoieties comprising P-halogen bonds are included within the definitionof activated phosphorous moieties. Unless stated otherwise specificallyin the specification, an activated phosphorous group is optionallysubstituted.

“Protected hydroxyl” refers to a hydroxyl moiety wherein the H has beenreversibly replaced with a protecting group. Protecting groups are wellknown in the art. In certain embodiments, a protected hydroxyl will bean ether (e.g., alkoxy, arlyalkyloxy or aryloxy). A non-limiting exampleof a protected hydroxyl is dimethoxytrityl ether. Other protectedhydroxyl moieties are well-known in the art. Unless stated otherwisespecifically in the specification, a protected hydroxyl group isoptionally substituted.

“Sulfhydrylalkyl” refers to an alkyl group comprising at least onesulfhydryl substituent. The one or more SH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, a sulfhydrylalkyl group is optionallysubstituted.

“Sulfhydrylalkylether” refers to an alkylether group comprising at leastone sulfhydryl substituent. The one or more —SH substituents may be on aprimary, secondary or tertiary carbon atom. Unless stated otherwisespecifically in the specification, a sulfhydrylalkylether group isoptionally substituted.

“Sulfonate” refers to the —OS(O)₂R_(a) group, wherein R_(a) is alkyl oraryl. Unless stated otherwise specifically in the specification, asulfonate group is optionally substituted.

“Thioalkyl” refers to a group of the formula —SR_(a) where R_(a) is analkyl group as defined above containing from one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup is optionally substituted.

“Thiophosphate” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH,O⁻, OR_(d), a phosphate group, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(d) is a counter ion (e.g., Na+ and the like) and provided that: R_(a)is S or R_(b) is S⁻ or SR_(d); or provided that R_(a) is S and R_(b) isS⁻ or SR_(d). Unless stated otherwise specifically in the specification,a thiophosphate group is optionally substituted.

“Thiophospho” refers to the —P(═R_(a))(R_(b))R_(c) group, wherein R_(a)is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH, O⁻,OR_(d), a phosphate group, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether, whereinR_(d) is a counter ion (e.g., Na+ and the like) and provided that: R_(a)is S or R_(b) is S⁻ or SR_(d); or provided that R_(a) is S and R_(b) isS⁻ or SR_(d). Unless stated otherwise specifically in the specification,a thiophospho group is optionally substituted.

“Thiophosphoalkyl” refers to the —P(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is—Oalkyl, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: R_(a) is S or R_(b) is S⁻ or SR_(d); or provided thatR_(a) is S and R_(b) is S⁻ or SR_(d). Unless stated otherwisespecifically in the specification, a thiophosphoalkyl group isoptionally substituted. For example, in certain embodiments, the alkylmoiety in a thiophosphoalkyl group is optionally substituted with one ormore of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether, which substituents are optionally substituted.

“Othiophosphoalkyl” is a thiophosphoalkyl group connected to theremainder of the molecule via an oxygen atom. Unless stated otherwisespecifically in the specification, an Othiophosphoalkyl group isoptionally substituted. For example, in certain embodiments, the alkylmoiety in an Othiophosphoalkyl group is optionally substituted with oneor more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate,phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether, which substituents are optionally substituted.

“Thiophosphoalkylether” refers to the —P(═R_(a))(R_(b))R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); andR_(c) is —Oalkylether, wherein R_(d) is a counter ion (e.g., Na+ and thelike) and provided that: R_(a) is S or R_(b) is S⁻ or SR_(d); orprovided that R_(a) is S and R_(b) is S⁻ or SR_(d). Unless statedotherwise specifically in the specification, a thiophosphoalkylethergroup is optionally substituted. For example, in certain embodiments,the alkylether moiety in a thiophosphoalkyl group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,phospho, thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents areoptionally substituted.

“Othiophosphoalkylether” is a thiophosphoalkylether group connected tothe remainder of the molecule via an oxygen atom. Unless statedotherwise specifically in the specification, an Othiophosphoalkylethergroup is optionally substituted. For example, in certain embodiments,the alkylether moiety in an Othiophosphoalkyl group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,phospho, thiophospho, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether, which substituents areoptionally substituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, alkoxy, alkylamino, alkylether, polyalkylether,hydroxylpolyalkylether, aminopolyalkylether, alkylenether, alkylphospho,alkyletherphospho, alkylthiophospho, alkyletherthiophospho, amidyl,thioalkyl, aryl, aryloxy, aralkyl, Oaralkyl, cyanoalkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl, heteroarylalkyl,hydroxylalkyl, aminoalkyl, hydroxylalkylether, phospho, phosphoalkyl,phosphoalkylether, phosphoramidite, activated phosphorous, protectedhydroxyl, sulfhydrylalkyl, sulfhydrylalkylether, sulfonate, thioalkyl,thiophospho, thiophosphoalkyl and/or thiophosphoalkylether) wherein atleast one hydrogen atom is replaced by a bond to a non-hydrogen atomssuch as, but not limited to: a halogen atom such as F, Cl, Br, and I; anoxygen atom in groups such as hydroxyl groups, alkoxy groups, and estergroups; a sulfur atom in groups such as thiol groups, thioalkyl groups,sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atomin groups such as amines, amides, alkylamines, dialkylamines,arylamines, alkylarylamines, diarylamines, N-oxides, imides, andenamines; a silicon atom in groups such as trialkylsilyl groups,dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilylgroups; and other heteroatoms in various other groups. “Substituted”also means any of the above groups in which one or more hydrogen atomsare replaced by a higher-order bond (e.g., a double- or triple-bond) toa heteroatom such as oxygen in oxo, carbonyl, carboxyl, and estergroups; and nitrogen in groups such as imines, oximes, hydrazones, andnitriles. For example, “substituted” includes any of the above groups inwhich one or more hydrogen atoms are replaced with —NR_(g)R_(h),—NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h),—NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g),—SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h).“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced with —C(═O)R_(g), (═O)OR_(g),(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). In the foregoing,R_(g) and R_(h) are the same or different and independently hydrogen,alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl.“Substituted” further means any of the above groups in which one or morehydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl,imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl,aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl group. In addition, each of the foregoing substituentsmay also be optionally substituted with one or more of the abovesubstituents.

“Fluorescent” refers to a molecule which is capable of absorbing lightof a particular frequency and emitting light of a different frequency.Fluorescence is well-known to those of ordinary skill in the art.

“Colored” refers to a molecule which absorbs light within the coloredspectrum (i.e., red, yellow, blue and the like).

A “linker” refers to a contiguous chain of at least one atom, such ascarbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof,which connects a portion of a molecule to another portion of the samemolecule or to a different molecule, moiety or solid support (e.g.,microparticle). Linkers may connect the molecule via a covalent bond orother means, such as ionic or hydrogen bond interactions.

For purposes of the present invention, the term “biomolecule” refers toany of a variety of biological materials, including nucleic acids,carbohydrates, amino acids, polypeptides, glycoproteins, hormones,aptamers and mixtures thereof. More specifically, the term is intendedto include, without limitation, RNA, DNA, oligonucleotides, modified orderivatized nucleotides, enzymes, receptors, prions, receptor ligands(including hormones), antibodies, antigens, and toxins, as well asbacteria, viruses, blood cells, and tissue cells. The visuallydetectable biomolecules of the invention (i.e., compounds of structure(I) having a biomolecule linked thereto) are prepared, as furtherdescribed herein, by contacting a biomolecule with a compound having areactive group that enables attachment of the biomolecule to thecompound via any available atom or functional group, such as an amino,hydroxy, carboxyl, or sulfhydryl group on the biomolecule.

The terms “visible” and “visually detectable” are used herein to referto substances that are observable by visual inspection, without priorillumination, or chemical or enzymatic activation. Such visuallydetectable substances absorb and emit light in a region of the spectrumranging from about 300 to about 900 nm. Preferably, such substances areintensely colored, preferably having a molar extinction coefficient ofat least about 40,000, more preferably at least about 50,000, still morepreferably at least about 60,000, yet still more preferably at leastabout 70,000, and most preferably at least about 80,000 M⁻¹ cm⁻¹. Thebiomolecules of the invention may be detected by observation with thenaked eye, or with the aid of an optically based detection device,including, without limitation, absorption spectrophotometers,transmission light microscopes, digital cameras and scanners. Visuallydetectable substances are not limited to those which emit and/or absorblight in the visible spectrum. Substances which emit and/or absorb lightin the ultraviolet (UV) region (about 10 nm to about 400 nm), infrared(IR) region (about 700 nm to about 1 mm), and substances emitting and/orabsorbing in other regions of the electromagnetic spectrum are alsoincluded with the scope of “visually detectable” substances.

For purposes of the invention, the term “photostable visible dye” refersto a chemical moiety that is visually detectable, as definedhereinabove, and is not significantly altered or decomposed uponexposure to light. Preferably, the photostable visible dye does notexhibit significant bleaching or decomposition after being exposed tolight for at least one hour. More preferably, the visible dye is stableafter exposure to light for at least 12 hours, still more preferably atleast 24 hours, still yet more preferably at least one week, and mostpreferably at least one month. Nonlimiting examples of photostablevisible dyes suitable for use in the compounds and methods of theinvention include azo dyes, thioindigo dyes, quinacridone pigments,dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole,quinophthalone, and truarycarbonium.

The visually detectable biomolecules of the invention are useful for awide variety of biochemical and biomedical applications in which thereis a need to determine the presence, location, or quantity of aparticular biomolecule. In another aspect, therefore, the inventionprovides a method for visually detecting a biomolecule, comprising: (a)providing a biological system with a visually detectable biomoleculecomprising the compound of structure (I) linked to a biomolecule; and(b) detecting the biomolecule by its visible properties. For purposes ofthe invention, the phrase “detecting the biomolecule by its visibleproperties” means that the biomolecule, without illumination or chemicalor enzymatic activation, is observed with the naked eye, or with the aidof a optically based detection device, including, without limitation,absorption spectrophotometers, transmission light microscopes, digitalcameras and scanners. A densitometer may be used to quantify the amountof visually detectable biomolecule present. For example, the relativequantity of the biomolecule in two samples can be determined bymeasuring relative optical density. If the stoichiometry of dyemolecules per biomolecule is known, and the extinction coefficient ofthe dye molecule is known, then the absolute concentration of thebiomolecule can also be determined from a measurement of opticaldensity. As used herein, the term “biological system” is used to referto any solution or mixture comprising one or more biomolecules inaddition to the visually detectable biomolecule. Nonlimiting examples ofsuch biological systems include cells, cell extracts, tissue samples,electrophoretic gels, assay mixtures, and hybridization reactionmixtures.

“Microparticle” refers to any of a number of small particles useful forattachment to compounds of the invention, including, but not limited to,glass beads, magnetic beads, polymeric beads, nonpolymeric beads, andthe like. In certain embodiments, a microparticle comprises polystyrene,such as polystyrene beads.

Embodiments of the invention disclosed herein are also meant toencompass all compounds of structure (I) being isotopically-labelled byhaving one or more atoms replaced by an atom having a different atomicmass or mass number. Examples of isotopes that can be incorporated intothe disclosed compounds include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H,¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I,and ¹²⁵I, respectively.

Isotopically-labeled compounds of structure (I) can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described below and in the followingExamples using an appropriate isotopically-labeled reagent in place ofthe non-labeled reagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted alkyl” means that thealkyl group may or may not be substituted and that the descriptionincludes both substituted alkyl groups and alkyl groups having nosubstitution.

“Salt” includes both acid and base addition salts.

“Acid addition salt” refers to those salts which are formed withinorganic acids such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Base addition salt” refers to those salts which are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asammonia, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. The present invention includes all solvates of the describedcompounds. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compounds of the invention may be truesolvates, while in other cases the compounds of the invention may merelyretain adventitious water or another solvent or be a mixture of waterplus some adventitious solvent.

The compounds of the invention, or their salts, tautomers or solvatesmay contain one or more asymmetric centers and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds. Various tautomeric forms of thecompounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0software naming program (CambridgeSoft). Common names familiar to one ofordinary skill in the art are also used.

As noted above, in one embodiment of the present invention, compoundsuseful as fluorescent and/or colored dyes in various analytical methodsare provided. The compounds have the following structure (I):

or a salt, stereoisomer or tautomer thereof, wherein:

R^(1a) and R^(1b) are each independently hydroxyl or alkoxy; and

R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r)and R^(2s) are each independently H, halo or -L¹-R³;

R³ is, at each occurrence, independently an analyte molecule or alkylsubstituted with one or more of hydroxyl, protected hydroxyl, amino,alkylamino, alkoxy, polyalkylether, hydroxylalkoxy, aminoalkoxy,hydroxylpolyalkylether, aminopolyalkylether, phosphate, thiophosphate,phospho, thiophospho, phosphoalkylether, Ophosphoalkyletherthiophosphoalkylether, Othiophosphoalkylether, phosphoramidite oractivated phosphorous; or R³ is a microparticle; and

L¹ is an optional linker moiety.

In some other embodiments, the compound has one of the followingstructures (Ia) or (Ib):

In more embodiments, the compound has one of the following structures(Ic) or (Id):

In still other embodiments, the compound has one of the followingstructures (Ie) or (If):

In some specific embodiments of the above, the compound has structure(If).

In various embodiments, R^(1a) or R^(1b) is hydroxyl. In otherembodiments, R^(1a) or R^(1b) is alkoxy. For example, in someembodiments each of R^(1a) and R^(1b) are alkoxy. In some of theforegoing embodiments, alkoxy is C₁-C₆alkoxy, such as methoxy.

In some of any of the foregoing embodiments, at least one of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j),R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) or R^(2s)is H.

In other of the foregoing embodiments, at least one of R^(2a), R^(2b),R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k),R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) or R^(2s) ishalo. In some more specific embodiments, at least one of R^(2a), R^(2b),R^(2c), R^(2d), R^(2e), R^(2f), or R^(2h) is halo, for example in someembodiments R^(2b) is halo. In some of any of the foregoing embodiments,halo is bromo.

In various other embodiments, at least one of R^(2a), R^(2b), R^(2c),R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l),R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) or R^(2s) is -L¹-R³. Forexample, in some embodiments at least one of R^(2a), R^(2b), R^(2c),R^(2d), R^(2e), R^(2f), R^(2g) is -L¹-R³. In more specific embodiments,R^(2b) is -L¹-R³.

In still other embodiments, R^(2b) is -L¹-R³ and R^(2a), R^(2c), R^(2d),R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l), R^(2m),R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) and R^(2s) are H.

In some embodiments, R³ is, at each occurrence, independently an analytemolecule or alkyl substituted with one or more of hydroxyl, protectedhydroxyl, amino, alkylamino, alkoxy, polyalkylether, hydroxylalkoxy,aminoalkoxy, hydroxylpolyalkylether, aminopolyalkylether, phosphate,thiophosphate, phospho, thiophospho, phosphoalkylether,thiophosphoalkylether, phosphoramidite or activated phosphorous; or R³is a microparticle. In some of the foregoing embodiments, thephosphoalkylether is an Ophosphoalkylether. In other of the foregoingembodiments, the thiophosphoalkylether is an Othiophosphoalkylether.

In some of the foregoing embodiments, R³ is, at each occurrence,independently alkyl substituted with one or more of hydroxyl, protectedhydroxyl, amino, alkylamino, alkoxy, polyalkyether, hydroxylalkoxy,aminoalkoxy, hydroxylpolyalkyether, aminopolyalkyether, phosphate,thiophosphate, phospho, thiophospho, phosphoalkylether,thiophosphoalkylether, phosphoramidite or activated phosphorous. Forexample, in some embodiments R³ is alkyl substituted with one or moresubstituents selected from hydroxyl, amino, trityl ether,phophoramidite, phospho, phosphoalkylether, phosphate and polyethyleneglycol.

In some of the foregoing embodiments, R³ is, at each occurrence,independently alkyl substituted with one or more of hydroxyl, protectedhydroxyl, amino, alkylamino, alkoxy, polyalkyether, hydroxylalkoxy,aminoalkoxy, hydroxylpolyalkyether, aminopolyalkyether, phosphate,thiophosphate, phospho, thiophospho, phosphoalkylether,Ophosphoalkylether, thiophosphoalkylether, Othiophosphoalkylether,phosphoramidite or activated phosphorous. For example, in someembodiments R³ is alkyl substituted with one or more substituentsselected from hydroxyl, amino, trityl ether, phophoramidite, phospho,phosphoalkylether, Ophosphoalkylether, phosphate and polyethyleneglycol.

In some embodiments, R³ is, at each occurrence, independently alkylsubstituted with one or more Ophosphoalkylether.

For example, in some embodiments of the above, R³ is, at eachoccurrence, independently alkyl substituted with one or morephosphoalkyether groups, such as Ophosphoalkyether groups, and the alkylether of the polyalkylether is a polyalkylether such as ethylene glycol.In some of these embodiments the alkylether of the phosphoalkylether issubstituted with a substituent selected from hydroxyl and a furtherphopshoalkylether moiety, such as a further Ophosphoalkyether moiety,which may be substituted with hydroxyl and/or amino.

In other embodiments, R³ is, at each occurrence, independently alkylsubstituted with two Ophosphoalkyether groups. In some of theseembodiments, the alkylether moiety is a polyalkylether, such as apolyethylene oxide. In some other of these embodiments, the alkyethermoieties are substituted with one or more substituents selected fromhydroxyl, amino and a further a further Ophosphoalkyether moiety, andthe alkylether of the further Ophosphoalkyether moiety is optionallysubstituted with hydroxyl and/or amino.

It is understood that some embodiments of the compound of structure (I)comprise two or more R³ groups. In such embodiments, the R³ groups maybe the same or different. In certain embodiments, the compound ofstructure (I) comprises a single R³ moiety. i.e., only one of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2i), R^(2j), R^(2k),R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) or R^(2s) is-L¹-R³.

In various other embodiments of any of the foregoing, R³ has one of thefollowing structures:

wherein:

n¹, n² and n³ are each independently an integer from 1 to 6; and

x and y are each independently an integer from 1 to 10.

In some of the above embodiments, n¹, n² and n³ are each 1. In otherembodiments, L¹ is absent and R³ connects directly to the remainder ofthe compound of structure (I).

In some other embodiments of the above, x is 5. In other embodiments, yis 5. In some more embodiments x and y are each 5.

In some different embodiments, R³ is an analyte molecule, such as abiomolecule. In certain embodiments R³ is a biomolecule selected fromnucleic acids, amino acids and polymers thereof (e.g., DNA,oligonucleotides, proteins, polypeptides, and the like).

In still other embodiments, R³ is a biomolecule, and the biomolecule isa nucleic acid, peptide, carbohydrate, lipid, enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer, antigen or prion.

In some different embodiments, R³ is an analyte molecule selected from adrug, vitamin and small molecule.

In some of the foregoing embodiments, the linker L¹ is present. Whenpresent, L¹ is conveniently selected to provide a covalent attachmentbetween R³ and the remainder of the compound of structure (I). ExemplaryL¹ moieites include, but are not limited to, O, N, S, P and alkylenebonds, and combinations thereof. Suitable L¹ groups are derivable by oneof ordinary skill in the art.

In other embodiments, L¹ is absent.

In some embodiments where L¹ is present, L¹ comprises a phosphate bondto the analyte molecule. In some of these embodiments, L¹ has thefollowing structure:

wherein n² and n³ are each independently an integer from 1 to 6. Invarious other embodiments, n¹, n² and n³ are each 1.

The structure of the compound of structure (I) is selected to optimizethe absorbance and or emission wavelengths. Accordingly, in variousembodiments the compound of structure (I) has a maximum absorbanceranging from about 468 nm to about 508 nm, for example from about 478 nmto about 498 nm. In other embodiments, the compound of structure (I) hasa maximum emission ranging from about 495 nm to about 525 nm, forexample, from about 495 to about 515 nm. For example, in certainembodiment the dyes have a maximum absorbance at about 490 nm and amaximum emission at about 505 nm.

In some more specific embodiments, the compound of structure (I) has oneof the following structures:

wherein R³ is an analyte molecule.

In various other embodiments, the invention provides an analyte moleculecomprising a covalent bond to a compound having the following structure(I′):

or a salt thereof, wherein:

R^(1a) and R^(1b) are each independently hydroxyl or alkoxy; and

R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r)and R^(2s) are each independently H, halo or -L¹-R³;

R³ is the analyte molecule; and

L¹ is an optional linker moiety,

wherein at least one of R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f),R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o),R^(2p), R^(2q), R^(2r) or R^(2s) is -L¹-R³.

The analyte molecule may be selected from any appropriate analytemolecules, including the analyte molecules described herein above.

Compositions comprising any of the foregoing compounds and one or morebiomolecules are provided in various other embodiments. In someembodiments, use of such compositions in analytical methods fordetection of the one or more biomolecules is also provided as describedin more detail below.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific choice set forth herein for aR^(1a), R^(1b), R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g),R^(2h), R^(2i), R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p),R^(2q), R^(2r), R^(2s), L¹ or R³ variable in the compounds of structure(I), as set forth above, may be independently combined with otherembodiments and/or variables of the compounds of structure (I) to formembodiments of the inventions not specifically set forth above. Inaddition, in the event that a list of choices is listed for anyparticular R or L group in a particular embodiment and/or claim, it isunderstood that each individual choice may be deleted from theparticular embodiment and/or claim and that the remaining list ofchoices will be considered to be within the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their salts by treatment with theappropriate inorganic or organic base or acid by methods known to oneskilled in the art. Salts of the compounds of the invention can beconverted to their free base or acid form by standard techniques.

The following Reaction Scheme illustrates exemplary methods of makingcompounds of this invention, i.e., compound of structure (I):

wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f),R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o),R^(2p), R^(2q), R^(2r), R^(2s), R³ and L¹ are as defined above.

It is understood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of structure (I) not specifically illustrated below by usingthe appropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

Reaction Scheme I illustrates an exemplary method for preparingcompounds of structure I. Referring to Reaction Scheme 1, compounds ofstructure A and B can be purchased or prepared by methods well-known tothose of ordinary skill in the art. Treatment of 2 equivalents of A witha strong base, such as n-butyl lithium, followed by reaction with Bresults in compounds of structure (I). Although Reaction Scheme Idepicts preparation of symmetrical (i.e., identical naphthyl groups)compounds of structure (I), it will be readily apparent to one ofordinary skill in the art that other, non-symmetrical, compounds ofstructure (I) can be prepared by similar methods (e.g., stepwisereaction of differently substituted napthyls).

Further, compounds of structure (I) obtained by the above methods can befurther modified to obtain different compounds of structure (I). Forexample, in certain embodiments the compounds of structure (I) compriseat least one -L¹-R³ moiety. In such embodiments, the -L¹-R³ moiety, orprecursor thereof, may be installed via any well-known method, such asSuzuki coupling. Analyte molecules (e.g., biomolecules) can be attachedvia an optional L¹ linker by any one of many common methods. Forexample, modification of the above scheme to include reactive groupscapable of forming covalent bonds with a functional group on an analytemolecule is one means for attaching analyte molecules. Reactive groupsinclude, but are not limited to activated phosphorus compounds (e.g.,phosphoramidites), activated esters, amines, alcohols, and the like.Methods for preparation of such compounds and reacting the same with ananalyte molecule to form a covalent bond are well-known in the art.

In some embodiments, the compounds of structure (I) comprise a covalentbond to an oligonucleotide. Such bonds may be formed by including aphosphoramidite moiety in the compound of structure (I) and reacting thesame with an oligomer (or phosphoramidite monomer) under standard DNAsynthesis conditions. DNA synthesis methods are well-known in the art.Briefly, two alcohol groups are functionalized with a dimethoxytrityl(DMT) group and a 2-cyanoethyl-N,N-diisopropylamino phosphoramiditegroup, respectively. The phosphoramidite group is coupled to an alcoholgroup, typically in the presence of an activator such as tetrazole,followed by oxidation of the phosphorous atom with iodine. Thedimethoxytrityl group can be removed with acid (e.g., chloroacetic acid)to expose the free alcohol, which can be reacted with a phosphoramiditegroup. The 2-cyanoethyl group can be removed after oligomerization bytreatment with aqueous ammonia.

Preparation of the phosphoramidites used in the oligomerization methodsis also well-known in the art. For example, a primary alcohol can beprotected as a DMT group by reaction with DMT-Cl. A secondary alcohol isthen functionalized as a phosphoramidite by reaction with an appropriatereagent such as 2-cyanoethyl N,N-dissopropylchlorophosphoramidite.Methods for preparation of phosphoramidites and their oligomerizationare well-known in the art and described in more detail in the examples.

In still other embodiments, the compounds are useful in variousanalytical methods. For example, in certain embodiments the disclosureprovides a method of staining a sample, the method comprising adding tosaid sample a compound of structure (I) in an amount sufficient toproduce an optical response when said sample is illuminated at anappropriate wavelength.

In yet other embodiments of the foregoing method, one of R^(2a), R^(2b),R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k),R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) or R^(2s) is-L¹-R³ where R³ is an analyte molecule such as a biomolecule. Forexample, in some embodiments the biomolecule is nucleic acid, amino acidor a polymer thereof (e.g., polynucleotide or polypeptide). In stillmore embodiments, the biomolecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion.

In yet other embodiments of the foregoing method, one of R^(2a), R^(2b),R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k),R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) or R^(2s) is-L¹-R³ where R³ is a microparticle. For example, in some embodiments themicroparticle is a polymeric bead or nonpolymeric bead.

In even more embodiments, said optical response is a fluorescentresponse.

In other embodiments, said sample comprises cells, and some embodimentsfurther comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishingthe fluorescence response from that of a second fluorophore havingdetectably different optical properties.

In other embodiments, the disclosure provides a method for visuallydetecting a biomolecule, comprising:

(a) providing a compound of structure (I), wherein one of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j),R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2r) or R^(2s) is-L¹-R³ where R³ is a biomolecule; and

(b) detecting the compound by its visible properties.

For example, in some embodiments the biomolecule is a nucleic acid,amino acid or a polymer thereof (e.g., polynucleotide or polypeptide).In still more embodiments, the biomolecule is an enzyme, receptor,receptor ligand, antibody, glycoprotein, aptamer or prion.

In other embodiments, a method for visually detecting a biomolecule isprovided, the method comprising:

(a) admixing any of the foregoing compounds with one or morebiomolecules; and

(b) detecting the compound by its visible properties.

As noted above, certain embodiments of the compounds of structure (I)comprise an analyte molecule (e.g., biomolecule) or a ligand attached(conjugated) thereto. Attachment may be, for example, by covalentbonding, ionic bonding, dated bonding, hydrogen bonding, and other formsof molecular bonding.

Several types of analyte molecules are suitable for conjugation to thecompounds of structure (I). For example, useful conjugated substrates ofthe invention include, but are not limited to, compounds of structure(I) comprising an analyte molecule attached thereto (also referred toherein as a “conjugated substrate”), the analyte molecule being selectedfrom antigens, small molecules, steroids, vitamins, drugs, haptens,metabolites, toxins, environmental pollutants, amino acids, peptides,proteins, photosensitizers, nucleotides, oligonucleotides, nucleicacids, carbohydrates, lipids, ion-complexing moieties and non-biologicalpolymers. In one exemplary embodiment, the conjugated substrate is anatural or synthetic amino acid, a natural or synthetic peptide orprotein, or an ion-complexing moiety. Exemplary peptides include, butare not limited to protease substrates, protein kinase substrates,phosphatase substrates, neuropeptides, cytokines, and toxins. Exemplaryprotein conjugates include enzymes, antibodies, lectins, glycoproteins,histones, alumin, lipoproteins, avidin, streptavidins, protein A,protein G, casein, phycobiliproteins, other fluorescent proteins,hormones, toxins, growth factors, and the like.

The point of attachment of the analyte molecule to the remainder of thecompound of structure (I) can and will vary depending upon theembodiment. Further, some embodiments include a linker (L¹) between theanalyte molecule and the remainder of the compound of structure (I),although use of the linker is optional and not required in allembodiments. It is also envisioned that the compound of structure (I)may comprise more than one analyte molecule. For example, two, three ormore than three analyte molecules may be conjugated to the naphthyland/or anthracene rings of compound (I).

Several methods of linking dyes to various types of analyte moleculesare well known in the art. For example, methods for conjugating dyes toan analyte molecule are described in R. Haugland, The Handbook: A Guideto Fluorescent Probes and Labeling Technologies, 9th Ed., 2002,Molecular Probes, Inc., and the references cited therein; and Brindley,1992, Bioconjugate Chem. 3:2, which are all incorporated herein byreference. By way of example, a compound of the disclosure may include acovalent bond to DNA or RNA via one or more purine or pyrimidine basesthrough an amide, ester, ether, or thioether bond; or is attached to thephosphate or carbohydrate by a bond that is an ester, thioester, amide,ether, or thioether. Alternatively, a compound of structure (I) may bebound to the nucleic acid by chemical post-modification, such as withplatinum reagents, or using a photoactivatable molecule such as aconjugated psoralen.

xThe compounds of the invention are useful in many applicationsincluding those described for other dyes in U.S. Pat. Nos. 7,172,907;5,268,486; and U.S. Patent Application Nos. 20040014981; and20070042398, all of which are incorporated herein by reference in theirentireties. For example, fluorescent dyes, such as those describedherein, may be used in imaging with techniques such as those based onfluorescence detection, including but not limited to fluorescencelifetime, anisotropy, photoinduced electron transfer, photobleachingrecovery, and non-radioactive transfer. The compounds of structure (I),as such, may be utilized in all fluorescent-based imaging, microscopy,and spectroscopy techniques including variations on such. In addition,they may also be used for photodynamic therapy and in multimodalimaging. Exemplary fluorescence detection techniques include those thatinvolve detecting fluorescence generated within a system. Suchtechniques include, but are not limited to, fluorescence microscopy,fluorescence activated cell sorting (FACS), fluorescent flow cytometry,fluorescence correlation spectroscopy (FCS), fluorescence in situhybridization (FISH), multiphoton imaging, diffuse optical tomography,molecular imaging in cells and tissue, fluorescence imaging with onenanometer accuracy (FIONA), free radical initiated peptide sequencing(FRIPs), and second harmonic retinal imaging of membrane potential(SHRIMP), as well as other methods known in the art.

Alternatively, the compounds of structure (I) can be used as markers ortags to track dynamic behavior in living cells. In this regard,fluorescence recovery after photobleaching (FRAP) can be employed incombination with the subject compounds to selectively destroyfluorescent molecules within a region of interest with a high-intensitylaser, followed by monitoring the recovery of new fluorescent moleculesinto the bleached area over a period of time with low-intensity laserlight. Variants of FRAP include, but are not limited to, polarizing FRAP(pFRAP), fluorescence loss in photo-bleaching (FLIP), and fluorescencelocalization after photobleaching (FLAP). The resulting information fromFRAP and variants of FRAP can be used to determine kinetic properties,including the diffusion coefficient, mobile fraction, and transport rateof the fluorescently labeled molecules. Methods for such photo-bleachingbased techniques are described in Braeckmans, K. et al., BiophysicalJournal 85: 2240-2252, 2003; Braga, J. et al., Molecular Biology of theCell 15: 4749-4760, 2004; Haraguchi, T., Cell Structure and Function 27:333-334, 2002; Gordon, G. W. et al., Biophysical Journal 68: 766-778,1995, which are all incorporated herein by reference in theirentireties.

Other fluorescence imaging techniques are based on non-radioactiveenergy transfer reactions that are homogeneous luminescence assays ofenergy transfer between a donor and an acceptor. Such techniques thatmay employ the use of the subject fluorescent dyes include, but are notlimited to, FRET, FET, FP, HTRF, BRET, FLIM, FLI, TR-FRET, FLIE, smFRET,and SHREK. These techniques are all variations of FRET.

The subject compounds may be used as biosensors such as a Ca²⁻ ionindicator; a pH indicator; a phosphorylation indicator, or an indicatorof other ions, e.g., magnesium, sodium, potassium, chloride and halides.For example, biochemical processes frequently involve protonation anddeprotonation of biomolecules with concomitant changes in the pH of themilieu. Substitution at the meso-position with different pH-sensitivegroups generates a variety of NIR fluorescent pH sensors with differentpKa's. To be effective, the substituents at the meso-position will be inextended π-conjugation with the fluorophore core to effect markedspectral changes in response to different pH environments.

Uses of the disclosed compounds are in no way limited to analyticalmethods. In various embodiments, the compounds are used as colorants ordyes in various applications. In this respect, the substituents on thecore anthracene and/or naphthalene moieties are not particularly limitedprovided the compound maintains its desired color and/or absorbanceand/or emission properties. Selection of appropriate substituents forthis purpose is within the skill of one of ordinary skill in the art.Accordingly, in some embodiments a compound useful as a dye or colorantis provided having the following structure (I″):

or a salt thereof, wherein R^(1a), R^(1b), R^(2a), R^(2b), R^(2c),R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l),R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) and R^(2s) are eachindependently H or a substituent, the substituent being selected basedon the desired color and or emission/absorbance properties of thecompound.

In certain embodiments of the compound of structure (I″) R^(1a) andR^(1b) are each independently hydroxyl or alkoxy; and R^(2a), R^(2b),R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k),R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r) and R^(2s) areeach independently H, halo or alkyl.

In some embodiments the compounds disclosed herein (e.g., (I), (I′)and/or (I″)) find utility as a dye or colorant in textiles, plastics,paints and/or safety devices (e.g., reflective materials, emergencylights, glow sticks, etc.) One of ordinary skill in the art will readilyrecognize other uses for the disclosed compounds.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES

General Methods

¹H NMR spectra were obtained on a JEOL 400 MHz spectrometer. ¹H spectrawere referenced against TMS. Reverse phase HPLC dye analysis wasperformed using a Waters Acquity UHPLC system with a 2.1 mm×50 mmAcquity BEH-C18 column held at 45° C. Mass spectral analysis wasperformed on a Waters/Micromass Quattro micro MS/MS system (in MS onlymode) using MassLynx 4.1 acquisition software. Mobile phase used forLC/MS on dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6mM triethylamine (TEA), pH 8. Phosphoramidites and precursor moleculeswere analyzed using an Agilent Infinity 1260 UHPLC system with a diodearray detector and High Performance Autosampler using an Aapptec©Spirit™ Peptide C18 column (4.6 mm×100 mm, 5 μm particle size).Molecular weights for monomer intermediates were obtained usingtropylium cation infusion enhanced ionization. Excitation and emissionprofiles experiments were recorded on a Cary Eclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic acid, pyridine, and THF were purchased fromAldrich. All other chemicals were purchased from Aldrich or TCI and wereused as is with no additional purification.

Example 1 SYNTHESIS OF 2-BROMO-9,10-BIS((6-METHOXYNAPHTHALEN-1-YL)ETHYNYL)ANTHRACENE (1)

A clean, dry 200 mL round bottom flask was flushed with nitrogen and dryTHF (40 mL). 1-ethynyl-6-methoxynapthalene (2.0 g, 10.9 mmol) was addedand the flask which was then cooled in an acetone dry ice bath under anitrogen atmosphere for 30 minutes. Upon cooling, nBuLi 2.5M in hexanes(4.4 mL, 10.9 mmol) was added dropwise, and the reaction was allowed tostir for 1 hr. 2-Bromoanthraquinone (1.05 g, 3.6 mmol) and ether (30 mL)were added, after which the reaction was allowed to warm to roomtemperature and stirred overnight. A solution of 0.1 M SnCl2 in 1M HCl(44 mL) was then added in one portion. After 2 hours, the reactionmixture was poured into methanol (200 mL), and the resulting slurry wasstirred for one hour. The red solid was isolated by filtration and driedunder vacuum (1.15 g, 51%).

The ¹H NMR spectrum of compound 1 is depicted in FIG. 1. The RP totaldiode array chromatogram at 215-500 nm of compound is depicted in FIG.2. The mass spectrum of compound 1 is depicted in FIG. 3.

Example 2 SYNTHESIS OF3-(9,10-BIS((6-METHOXYNAPHTHALEN-1-YL(ETHYNYL)ANTHRACEN-2-YL)-2-((BIS(4-METHOXYPHENYL)(PHENYL)METHOXY)METHYL)PROPAN-1-OL(2)

A 250 mL round bottom flask fitted with a condenser was purged withnitrogen and dry THF (50 mL) followed by addition of2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)prop-2-en-1-ol (2.2 g,5.6 mmol). 9-BBN 0.5 M in THF (30.7 mL, 14.0 mmol) was added viasyringe, and the reaction was heated to reflux for 12 hrs. Afterallowing the reaction to cool to room temperature, 3M K₂CO₃ (2.4 ml) anddry THF (150 mL) were added. Compound 1 (1.74 g, 2.8 mmol) andPdCl₂(dppf) (0.41 g, 0.56 mmol) were added, and the solution wasrefluxed for 6 hrs and allowed to cool to room temperature over twohours. The reaction mixture was poured into CH₂Cl₂ (300 mL) and washedwith H₂O (300 mL). The aqueous layer was back extracted with additionalCH₂Cl₂ (100 mL). The combined organic layers were washed with sat. NaCl(300 mL), dried over Na₂SO₄, and concentrated in vacuo to a viscous gum.The isolated crude product wash then purified by silica gel columnchromatography eluting with a gradient of CH₂Cl₂:hexanes (100:0v/v)-(0:100 v/v) give 3 as a red solid (1.0 g, 35%). The ¹H NMR spectrumof compound 2 is depicted in FIG. 4. The total diode array chromatogramat 215-500 nm of compound 2 is depicted in FIG. 5. The mass spectrum ofcompound 2 is depicted in FIG. 6.

Example 3 SYNTHESIS OF3-(9,10-BIS((6-METHOXYNAPHTHALEN-1-YL)ETHYNYL)ANTHRACEN-2-YL)-2-((BIS(4-METHOXYPHENYL)(PHENYL)METHOXY)METHYL)PROPYL2-CYANOETHYL DIISOPROPYLPHOSPHORAMIDITE (3)

A dry 100 mL round bottom flask was purged with nitrogen, followed byaddition of CH₂Cl₂ (20 mL) and compound 3 (0.20 g, 0.21 mmol).N,N-Diisopropylethylamine (0.18 mL, 10.7 mmol) and 2-cyanoethyldiisopropychlorophosphoramidite (0.15 mL, 0.6 mmol) were added viasyringe. After 1 hour of stirring at room temperature, the reaction wasdetermined to be complete by TLC analysis. The crude reaction mixturewas then concentrated in vacuo to a dark gum. The residue was dissolvedin acetonitrile and concentrated to dryness and used without furtherpurification for dye assembly.

Example 4 SYNTHESIS OF OLIGONUCLEOTIDE DYES

Compound 3 was used to prepare further dye compounds on an AppliedBiosystems 394 DNA/RNA synthesizer on a 1 μmol scale. The compoundscomprised a 3′-phosphate group. Dyes were synthesized directly on CPGbeads or on polystyrene solid support. The dyes were synthesized in the3′ to 5′ direction by standard solid phase DNA methods. Coupling methodsemployed standard β-cyanoethyl phosphoramidite chemistry conditions. Allphosphoramidite monomers were dissolved in acetonitrile/dichloromethane(0.1 M solutions), and were added in successive order using thefollowing synthesis cycles: 1) removal of the 5′-dimethoxytritylprotecting group with dichloroacetic acid in toluene, 2) coupling of thenext phosphoramidite with activator reagent in acetonitrile, 3)oxidation with iodine/pyridine/water, and 4) capping with aceticanhydride/1-methylimidizole/acetonitrile. The synthesis cycle wasrepeated until the 5′-oligofloroside was assembled. At the end of thechain assembly, the monomethoxytrityl (MMT) group or dimthoxytrityl(DMT) group was removed with dichloroacetic acid in dichloromethane ordichloroacetic acid in toluene. The dyes were cleaved from the solidsupport using concentrated aqueous ammonium hydroxide at roomtemperature for 2-4 hours. The product was concentrated in vacuo andSephadex G-25 columns were used to isolate the main product, which giveRP-HPLC traces as shown in FIG. 7 (compound 5). FIG. 8 shows massspectral data for dye prepared according to the above procedures(compound 5). Compound 4 was prepared according to the above generalprocedure by coupling compound 3 to the support followed by standardcleaveage and deprotection.

Structures, spectral properties and molecular weights (MW) determined byelectrospray mass spectrometry for exemplary compounds are presented inTable 1.

TABLE 1 Representative intermediates and oligonucleotide dye sequencesand their observed masses and optical properties. Calcu- Compound latedλmax, λmax, Name Description Mass Observed Mass abs em compound 45′-a6-3′ 706.7 707.6 490 505 compound 5 5′-NH3- 1563.9 1564.3 490 505(heg)-a6- (heg)-3′ compound 2 DMT- 929.1  930.1; 1020.1 488 508 alcohol(mass + Tr) Intermediate compound 1 brominated 617.5 618.2; 709.8 488508 Intermediate (mass + Tr) Code Desc. a6 Anthracene core NH35′Amino-5-modifier (heg) hexaethylene glycol spacer DMT4,4′-Dimethoxytrityl functional group Tr Tropylium cation

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification, includingU.S. provisional patent application Ser. No. 61/928,147, filed Jan. 16,2014, are incorporated herein by reference, in their entireties to theextent not inconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A method of staining a sample, the methodcomprising adding to the sample an analyte molecule in an amountsufficient to produce an optical response when said sample isilluminated at an appropriate wavelength, wherein: the analyte moleculehas the following structure (I′):

or a salt thereof, wherein: R^(1a) and R^(1b) are each independentlyhydroxyl or alkoxy; and R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f),R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o),R^(2p), R^(2q), R^(2r), R^(2s) and R^(2t) are each independently H, haloor -L¹-R³; R³ is, at each occurrence, independently a nucleic acid,peptide, carbohydrate, lipid, enzyme, receptor, receptor ligand,antibody, glycoprotein, aptamer, antigen, prion, drug, vitamin or smallmolecule; and L¹ is an optional linker moiety, wherein at least one ofR^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r),R^(2s) or R^(2t) is -L¹-R³.
 2. The method of claim 1, wherein saidoptical response is a fluorescent response.
 3. The method of claim 1,wherein said sample comprises cells.
 4. The method of claim 3 furthercomprising observing said cells by flow cytometry.
 5. The method ofclaim 4, further comprising distinguishing the fluorescence responsefrom that of a second fluorophore having detectably different opticalproperties.
 6. A method for visually detecting an analyte molecule, themethod comprising: (a) providing the analyte molecule; and (b) detectingthe analyte molecule by its visible properties wherein: the analytemolecule has the following structure (I′):

or a salt thereof, wherein: R^(1a) and R^(1b) are each independentlyhydroxyl or alkoxy; and R^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f),R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o),R^(2p), R^(2q), R^(2r), R^(2s) and R^(2t) are each independently H, haloor -L¹-R³; R³ is, at each occurrence, independently a nucleic acid,peptide, carbohydrate, lipid, enzyme, receptor, receptor ligand,antibody, glycoprotein, aptamer, antigen, prion, drug, vitamin or smallmolecule; and L¹ is an optional linker moiety, wherein at least one ofR^(2a), R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i),R^(2j), R^(2k), R^(2l), R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r),R^(2s) or R^(2t) is -L¹-R³.
 7. A method for visually detecting abiomolecule, the method comprising: (a) admixing a compound with one ormore biomolecules; and (b) detecting the compound by its visibleproperties wherein: the compound has the following structure (I):

or a salt, stereoisomer or tautomer thereof, wherein: R^(1a) and R^(1b)are each independently hydroxyl or alkoxy; and R^(2a), R^(2b), R^(2c),R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j), R^(2k), R^(2l),R^(2m), R^(2n), R^(2o), R^(2p), R^(2q), R^(2r), R^(2s) and R^(2t) areeach independently H, halo or -L¹-R³; R³ is, at each occurrence,independently the biomolecule, an alkyl substituted with one or more ofhydroxyl, protected hydroxyl, amino, alkylamino, alkoxy, polyalkyether,hydroxylalkoxy, aminoalkoxy, hydroxylpolyalkyether, aminopolyalkyether,phosphate, thiophosphate, phospho, thiophospho, phosphoalkylether,Ophosphoalkylether, thiophosphoalkylether, Othiophosphoalkylether,phosphoramidite or activated phosphorous; or R³ is a microparticle; andL¹ is an optional linker moiety.
 8. The method of claim 2, wherein saidsample comprises cells.